The primary objective of this research proposal is to identify how compartmentalization of thioredoxin (TRX) in the cell affects its function. The specific aims of this project are to identify where TRX localizes under conditions of oxidative stress, and how this localization affects gene regulation. The objectives of the grant proposal will be achieved in four distinct parts. First, we plan to show that TRX preferentially accumulates in the nucleus during oxidative stress. Second, we will manipulate the TRX gene in order to direct it to either localize in the nucleus or be secreted. We will construct TRX fusion plasmids that contain either TRX attached to a nuclear localization sequence or TRX attached to a secretory sequence. We will then compare differences in NF-kappa B- and AP-1 dependent promoter activity in cells overexpressing nuclear TRX compared with cells overexpressing secreted TRX. This will help establish the functional role of TRX compartmentalization. Third, we will establish that TRX localization is important for modulating interleukin-8 (IL-8) gene activity. Forth, using similar TRX expression plasmids as constructed for specific aim two, we will examine the effects of nucleus-directed TRX versus secreted TRX in mice exposed to ozone. Using this in vivo model, we plan to confirm the findings we obtained in our in vitro studies. The principal investigator's immediate research goals are to better understand the signaling mechanisms involved in airway epithelial cells in response to oxidative stress. Consistent with this objective, he currently has received funding for a three-year project examining the signaling mechanisms responsible for up-regulating IL-8 in airway epithelial cells exposed to cigarette smoke. It is the plan of the principle investigator to use this career development award as an excellent mechanism to transition into an independent investigator over the next five years. It is his hope that this grant funding will provide him with the protected research time needed in order to obtain the experience and publications needed to make him a competitive candidate for R01 funding.